A coomassie blue-binding assay for the microquantitation of immobilized proteins

ANALYTICAL BIOCHEMISTRY 148, 533-541 (1985)

A Coomassie Blue-Binding Assay for the Microquantitation of Immobilized Proteins

HASSAN AHMADAND M. SALEEMUDDIN

Biochemistry Division, Department of Chemistry, Aligarh Muslim University, Aligarh-202001, Uttar Pradesh, India

Received December 13, 1984

A sensitive assay procedure for the determination of microgram quantities of immobilized proteins is described. The procedure is based on the property of Coomassie blue G-250 to bind strongly yet reversibly to proteins. The assay involves incubation of the immobilized protein with a solution containing 0.1% Coomassie blue, 10% acetic acid, and 25% isopropyl alcohol in distilled water at room temperature followed by washing off of the unbound dye. The protein-bound dye is eluted with methanolic NaOH, acidified, and the absorbance is measured at 605 nm. The assay is highly reproducible and several proteins immobilized on various matrices could be conveniently assayed. Protein values determined by the dye-binding assay showed good agreement with those obtained by other procedures. o 1985 Academic PISS, I~C.

KEY WORDS: protein assay; immobilized proteins; Coomassie blue G-250.

Immobilized enzymes have numerous ac- tual and potential applications (1,2). In addition the study of immobilized enzymes yields valuable informations on the in vivo behavior of the particle-associated enzymes (3). Since artificial immobilization of enzymes usually results in the loss of varying amounts of the biological activity, accurate estimation of the immobilized protein becomes an essential part of the characterization of immobilized enzymes. A variety of methods are available for the estimation of proteins in the insolubilized preparations. These include measurement of the difference between added protein and protein recovered after immobilization (4), amino acid analysis after acid hydrolysis (5), assay of tightly bound pros- thetic groups or metal ions (6), analysis of elements like nitrogen and sulfur (7-9), and assay of ninhydrin-positive material after pronase digestion (10). The more convenient among these are the assays based upon acid hydrolysis followed by amino acid analysis and determination of the difference between protein added to the reaction mixture and that washed out of the conjugate after the

complexing process. While the first method requires special instrumentation the latter may not yield very accurate results since several proteins may bind nonspecifically to matrices and necessitate extensive washing for their complete removal. The washings may therefore contain too low a concentration of protein for estimation by most methods of protein analysis.

A sensitive and simple assay based on the binding of Coomassie blue G-250 with insolubilized protein is described. In this assay insolubilized proteins are incubated with excess dye and the unbound dye is washed off by repeated washing of the matrix either with distilled water or with a mixture of acetic acid and isopropyl alcohol. The dye bound to the insoluble protein is eluted with methanolic NaOH and acidified and absorbance is measured at 605 nm. The assay is highly reproducible and could be applied to several proteins coupled to various matrices.

MATERIALS AND METHODS

Hemoglobin, bovine serum albumin, ovalbumin, urease, cytochrome c, glass beads,

533 0003-2697185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

534 AHMAD AND SALEEMUDDIN

Coomassie blue G-250, and AE-cellulose were obtained from Sigma Chemical Company, St. Louis, Missouri. Sephadex G-100 and Sepharose 4B were purchased from Phar- macia Fine Chemicals, Uppsala, Sweden. Al- dolase, CM-cellulose, and 3-aminopropryltriethoxysilane were the products of Serva, West Germany and E. Merck, Darmstadt, West Germany, respectively. Other chemicals used were of analytical grade or the best grade available.

Preparation of immobilized proteins. The procedure described by Porath et al. (11) was followed for the insolubilization of proteins on Sepharose or Sephadex. Sepharose and Sephadex were washed extensively and activated by reaction with cyanogen bromide (100 mg/g gel). The slurry was stirred at 4°C for 10 min. The gel was finally washed with cold water and 0.1 M sodium bicarbonate, pH 8.5. The activated moist gel was placed in the appropriate protein solution and stirred in cold for 18 h. The unbound protein was separated from the gel matrix by centrifugation and the supematant retained for the determination of protein. In order to obtain the required level of protein immobilization the matrix was activated with excess cyanogen bromide (200 mg/g gel) prior to the addition of protein. It was thus possible to obtain insoluble preparations containing the desired amounts of protein since essentially all the added protein was coupled to the activated matrix under these conditions. A related procedure described by Axen and Emback (4) was used for the coupling of proteins on to cellulose.

Proteins were immobilized on AE-cellulose as described by Glassmeyer and Ogle (12). AE-cellulose was first activated by suspension in 0.5 N NaOH for 30 min. After the excess NaOH was washed off, the matrix was resus- pended in 0.5 M phosphate buffer, pH 7.0, and glutaraldehyde was added to a final concentration of 10% (v/v). The solution was gently stirred at room temperature for 2 h. Excess glutaraldehyde was removed by washing with 0.1 M phosphate buffer. Each gram

of the washed cellulose matrix was suspended in 10 ml of protein solution and gently stirred in cold for 12 h. Unreacted aldehydic groups were neutralized by reaction with 0.0 1% ethanolamine solution in 0.1 M phosphate buffer, pH 7.0.

Hemoglobin was covalently coupled to glass beads using a slight modification of the procedure described by Jacobson et al. (13). Two grams of acid-washed glass beads (75- 150 pm) were washed once with water and twice with acetone and dried at 100°C for 16-20 h. The dried beads were kept in a capped glass tube and 8.0 ml of 3.5% 3- aminopropryltriethoxysilane in anhydrous toluene was added. The glass beads were incubated at 85-90°C for 7 days with occasional shaking. The beads were subsequently washed three times with toluene and then with acetone and dried at room temperature. The aminopropyl glass beads were then treated with 10% (v/v) glutaraldehyde at room temperature for 4 h. Excess glutaraldehyde was finally removed by washing and the beads were suspended in hemoglobin solution and stirred for 20 h in cold.

Preparation of standard dye reagent. Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 100 ml solution composed of 10% (v/v) glacial acetic acid and 25% (v/v) isopropyl alcohol in distilled water. The mixture was stirred for 1 h using a magnetic stirrer, filtered through a Whatman No. 1 filter paper, and stored in a stoppered bottle. The dye reagent was highly stable and could be stored at room temperature for over 2 months.

Standard protein solutions. For the preparation of calibration curves, standard solution of BSA, ovalbumin, and cytochrome c were prepared spectrophotometrically using the respective standard extinction values ( 14- 16). The concentration of aldolase was determined after dialysis of the commercial ammonium sulfate suspension, by the method of Bradford ( 17). Solutions (0.1%) of hemoglobin and urease were prepared gravimetri- tally in 0.1 M phosphate buffer, pH 7.0.

COOMASSIE BLUE PROTEIN ASSAY 535

Preparation of calibration curve. The procedure employed for the preparation of standard calibration curves for the quantitation of insolubilized protein was based on that described by Esen (18). Standard protein solution (l-l 5 ~1) containing l-20 pg of protein was spotted at the center of 25 X 25- mm Whatman No. 1 filter paper squares with the help of a 5-~1 Hamilton syringe. The protein was fixed by placing the filter squares in 20% trichloroacetic acid (TCA)’ solution for 15 min. The filter squares were treated with the standard dye reagent for 30 min at room temperature. Approximately 100 ml of dye reagent in a 250-ml beaker was used for staining 20-30 filter squares. Unspotted filter squares were similarly pro- cessed to correct for any background value of nonspecific dye adsorption on the filter squares. After extensive washing of the unbound dye with distilled water the protein spots were cut out and placed in glass centrifuge tubes and eluted with methanolic NaOH (0.1 N NaOH in 20% water and 80% methanol). After complete elution of dye the filter papers were rinsed with methanolic NaOH and removed from the tubes. The solution was made up to 3.0 ml with methanolic NaOH and acidified by adding 0.1 ml of 4 N HCI, and the absorbance was measured at 605 nm in a Bausch and Lomb Spectronic 20 spectrophotometer. A 2.5-cm* area of the paper containing no bound protein adsorbed a small quantity of the dye, which on elution gave an absorbance not exceeding an OD of 0.05 under these conditions.

Siliconization of glassware. Detergent- washed glass centrifuge tubes were soaked in 5% solution of dichlorodimethylsilane in chloroform for 10 min at room temperature. After they were rinsed with water several times the tubes were kept at 100°C overnight.

Iron estimation. Iron was estimated by the modification of the original procedure of Wong (19) as described by Oser (20). Heme-

I Abbreviations used: TCA, trichloroacetic acid; BSA. bovine serum albumin.

containing proteins (4 to 50 mg) in a total volume of 1.0 ml were mixed with 1.0 ml concentrated H2S04 and the contents vigor- ously mixed. Saturated potassium persulfate (0.2 ml) was then added and the solution was again thoroughly mixed and diluted to 3.0 ml with distilled water. Sodium tungstate (1 .O ml, 0.025%) was subsequently added to precipitate the protein. To 2.0 ml of the supernatant obtained after centrifugation was added 0.6 ml of 3 N potassium thiocyanate and the color was read at 550 nm. Ferric ammonium sulfate was used for the preparation of standard curve. Where required the iron concentration was converted into hemoglobin or cytochrome c concentration with the help of separate calibration curves. Cali- bration curves were constructed by simulta- neously determining the protein concentrations (17) and iron concentration in various aliquots of the standard protein solution.

RESULTS

Standard dye-binding assay of insolubilized proteins. A 0. l-ml suspension of the insoluble matrices containing l-20 pg bound proteins was mixed and incubated with 1 .O ml of the standard dye reagent in glass centrifuge tubes for 30 min at room temperature. The tubes were subjected to occasional shaking. The insolubilized preparations were subsequently separated from the unbound dye by centrifugation at 3000 rpm for 10 min and washed several times with the wash solution. Distilled water could be used for washing off the excess dye with matrices other than cellulose. Cellulosic supports nonspecifically adsorb significant amounts of Coomassie blue. It was therefore essential to wash cellulosic matrices with a wash solution composed of 10% (v/v) glacial acetic acid and 25% (v/v) isopropyl alcohol instead of water to eliminate the unbound dye. Five to six suspensions and centrifugations with 5 ml of wash solution were generally sufficient to remove the nonspecifically bound dye. It was extremely important to prevent any loss of the insoluble


material during aspiration of the dye reagent or the wash solution. The protein-bound dye was eluted from the washed matrix by adding 3.0 ml of methanolic NaOH (0.1 N NaOH in 20% water and 80% methanol) and vor- texing the tubes. The supernatant was de- canted carefully after centrifugation at slow speed and 0.1 ml of 4 N HCl was added. Absorbance of the solution was measured at 605 nm in a Bausch and Lomb Spectronic 20 spectrophotometer. Alternatively the dye eluted in methanolic NaOH could be acidified prior to separation of the insoluble matrix. The unreacted matrices and those activated and subsequently blocked with appropriate reagent did not bind appreciable amounts of dye under the experimental conditions. It was therefore not essential to run control samples containing matrices without bound proteins. Coomassie blue has a much higher extinction in apolar solvents (21). The sensitivity of the assay is therefore far greater than the assays in which shift in the absorp- tion of Coomassie blue, as a result of binding to protein, is measured in aqueous solutions ( 17,22-24).

A linear relationship existed between the amount of insoluble protein taken and the quantity of dye bound by the preparation, both in the case of Sepharose-bound hemoglobin and in the case of cytochrome c (Fig. 1). Similar linearity was also observed when hemoglobin and cytochrome c were fixed on filter paper and their dye-binding capacities measured. As evident from the figure the dye-binding capacities of Sepharose-bound hemoglobin and hemoglobin fixed on filter paper were almost indistinguishable. Similar results were obtained with cytochrome c. Cytochrome c, however, bound relatively higher amounts of the dye as compared to hemoglobin.

In view of the tendency of the dye to adhere to the walls of the glass tubes (17) it was extremely important to use thoroughly washed glassware for the dye-binding assay. Dye adhering to the walls of improperly

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FIG. 1. Response curves of the soluble and insoluble proteins by the dye-binding assay. Sepharose-bound hemoglobin and cytochrome c were used. The protein concentration of the preparations were calculated from the concentration of iron with the help of a calibration curve as described in the text. Varing aliquots of the insolubilized hemoglobin or cytochrome c were incubated with the dye reagent and washed, and the absorbance of the eluted dye was measured as described in the text. Appropriate amounts of the soluble proteins were also spotted on filter paper squares and fixed with TCA, and the dye-binding capacity was determined. Hemoglobin, fixed on filter paper (0) or Sepharose bound (0); cytochrome c, fixed on filter paper (m) or Sepharose bound (0). Values represent the means of at least three independent experiments performed in duplicate. Deviations from the mean values in the case of the insoluble preparation are indicated by bars.

cleaned glassware gets readily eluted with methanolic NaOH, leading to a marked overestimation. The problem can be best overcome by siliconization of the glasswares. Siliconized glass tubes were found to bind insignificant quantities of dye under the experimental conditions.

Eflect of immobilized protein concentration on the binding of Coomassie blue. Sepharose matrix containing varying concentrations of immobilized hemoglobin was prepared as described earlier. Aliquots of gel containing varying amounts of hemoglobin were analyzed for iron (20) and for protein content by the dye-binding assay. As shown in Fig. 2 good agreement was obtained between protein


ug IRONlgm MATRIX

FIG. 2. Effect of immobilized protein concentration on the dye-binding assay. Varying amounts of hemoglobin were immobilized per gram of Sepharose 4B as described in the text. Quantities of immobilized protein were determined by the dye-binding method (0). The values were also calculated on the basis of iron estimation (0). Values are the means of two closely agreeing experiments performed in duplicate. The maximum deviation from the mean values was in no case greater than 4%.

values obtained by the dye-binding assay and those calculated using iron values over the range investigated.

Applicability of the dye-binding assay to proteins insolubilized on various supports. Hemoglobin immobilized on various insoluble supports was used for this study. As shown in Table 1 good correlation existed between the values of protein coupled to cellulose, AE-cellulose, Sephadex G- 100, and nonporous glass beads calculated by dye- binding assay and iron estimation. The dye- binding assay could also be applied when protein was noncovalently bound to CM- cellulose. In this case the amount of protein adsorbed to the ion exchanger could also be determined after elution from the matrix with 0.25 M Tris-HCl, pH 10.2. The values of protein in the eluate as determined by Bradford’s assay (17) were comparable with those obtained by the dye-binding assay.

Application of dye-binding assay to various immobilized proteins. Six different proteins

TABLE I

APPLICATION OF THE DYE-BINDING ASSAY TO

HEM~CLOBIN IMMOBILIZED ON VARIOUS SUPFQRTS

mg protein immobilized/g matrix”

Dye-binding IrOn Bradford assay

snppon assay estimation after elution

Cellulose 8.9 10.2 -

AE-Cellulose 18.2 19.8 -

Sephadex G-100 12.5 11.2 -

Glass beads 4.9 5.7 -

CM-cellulose 8.5 8.0 1.6

a Values represent the mean of two experiments performed in duplicate. The maximum deviation from the mean values was in no case greater than 6%.

immobilized on Sepharose 4B as described by Porath et al. (11) were used for this study. A comparison was made of quantities of immobilized proteins as determined by the dye-binding method as well as those obtained by subtracting the amount of protein remaining after immobilization from that added originally to the activated matrix. As shown in Table 2, for all the proteins investigated the values of immobilized proteins calculated

TABLE 2

COMPARISON OF THE DYE-BINDING PROTEIN ASSAY

OF IMMOBILIZED PROTEINS WITH OTHER

ASSAY PROCEDURES

mg prolein immobiiized/g Sepharose’

Protein

Dye- binding

-Y

Difference

between added and

unimmobilized protein

Iron estimation

Ovalbumin 12.7 18.4 -

BSA 16.1 19.0 -

IJE4.W 5.5 7.1 -

Aldolase 8.5 11.8 -

Hemoglobin 25.6 31.2 24.1 Cytochrome c Il.1 15.6 10.1

a Each value represents the mean of at least two experiments perfornted in duplicate. The maximum deviation from the mean values was in no case greater than 6%.


by the two procedures differed significantly. The values calculated by the latter procedure were significantly higher than those determined by the dye-binding assay. For hemoglobin and cytochrome c, the amounts of protein insolubilized were also calculated by iron estimation and a better agreement between these values and those obtained by dye-binding assay was observed. Separate calibration curves were prepared for the determination of the quantity of each immobilized protein investigated.

Repetitive quantitation of single samples of insolubilized protein. Sepharose 4B matrix containing 10.2 mg bound BSA/g gel was used in this study. The dye-binding assay was applied to four aliquots of Sepharose containing different amounts of bound BSA. Subsequent to each assay the insolubilized preparations were thoroughly washed and reincubated with fresh dye reagent. Washing of the unbound Coomassie blue and elution of the protein-bound dye was performed as in the standard assay described earlier. It is evident from Table 3 that the same sample of insolubilized protein could be repeatedly

TABLE 3

APPLICABILITY OF THE DYE-BINDING ASSAY FOR THE REPETITIVE QUANTITATION OFSINGLESAMPLES

~FIN~~LuBIL~~EDPRoTEIN

Absorbance at 605 nmb A-Y no.’ Sample: 1 2 3 4

1 0.26 0.45 0.7 1 0.85 2 0.25 0.44 0.71 0.85 3 0.25 0.44 0.71 0.83 4 0.25 0.44 0.69 0.83 5 0.25 0.43 0.69 0.83

“Assay no. indicates the number of times the dye- binding assay was applied on the same sample of Se- pharose-bound BSA.

b Values represent means of at least three independent experiments performed in duplicate. The maximum deviation from the mean values was in no case greater than 4%.

assayed for protein, and the values obtained showed excellent agreement.

Efect of amino groups modification on the dye-binding method. It is generally believed that the side-chain amino groups of proteins are crucial for the binding of Coomassie blue (18,25,26). We have therefore investigated the effect of amino group modification by glutaraldehyde (27) on the dye-binding assay. A 1% solution of BSA was incubated with a final concentration of 0.4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, at room temperature. The reaction was terminated at the indicated time intervals by the addition of sodium bisulfite. Amino groups were quantitated by the trinitrobenzenesulfonic acid procedure (28). It is clear from Fig. 3 that about 70% of the free amino groups were modified when the crosslinking reaction was performed for two hours. However, binding of Coomassie blue by the crosslinked protein, either measured in solution (17) or after fixing on filter paper (18) exhibited only a slight decrease, even when 70% of the total amino groups of the protein were modified.

DISCUSSION

Coomassie brilliant blue and several other dyes that strongly bind to proteins with rea- sonable specificity are widely used for staining and semiquantitative determination of protein on chromatograms. More recently several protein assays based on the binding of dyes to protein in solution have also been described (17,22-24,29,30). The dye-binding assays of- fer several advantages (3 l-38), and hence are finding increasing applications in research and clinical laboratories. Data presented in this manuscript show that the Coomassie blue G-250-binding assay can be conveniently used for the sensitive measurement of proteins insolubilized on several supports.

Figure 1 shows that a linear relationship exists between the amount of dye bound and the amount of immobilized hemoglobin or


I I I 0.5 1.0 1.5 2.0 2.5 30

TIME IHR)

FIG. 3. Effect of amino-groups modification on the binding of Coomassie blue. A 1% solution of BSA in 0.1 M phosphate buffer was incubated with a final concentration of 0.4% glutaraldehyde at room temperature. The reaction was stopped at the indicated time intervals by the addition of sodium bisulfite. After extensive dialysis of the protein, suitable aliquots of the samples were analyzed for amino groups by the trinitroknzenesulfonic acid procedure (0) and for dye binding by the procedure described by Bradford (0) or Esen (0). Values represent the means of three closely agreeing experiments performed in duplicate. The maximum deviation from the mean values was in no case greater than 3%.

cytochrome c taken over a wide range. A similar increase in the amount of dye bound as a function of the amount of insolubilized protein was also observed with several other proteins. Cytochrome c, as evident from the figure, bound higher amounts of Coomassie blue than did hemoglobin, in agreement with the earlier observations ( 17,2 1,39). Significant variations in the dye-binding capacities of other proteins have also been reported (17,39- 41). It was essential, therefore, to prepare individual calibration curves for the quantitation of each immobilized protein. In view of the high sensitivity of the dye-binding assay, however, less than 1 mg protein is sufficient for the preparation of the standard curve (Fig. 1). The high sensitivity of the assay system is the result of the high molar extinction coefficient of Coomassie blue in polar solvents (21), and the sensitivity of the

assay could be further increased by eluting the protein-bound dye in smaller volumes of methanolic NaOH (21). The dye-binding capacity of protein was not affected even when the concentration of matrix-bound protein was raised several fold (Fig. 2), suggesting lack of any steric hindrence in the binding of the dye under these conditions.

Data presented in Table 1 show that the nature of matrix does not influence the binding of dye to the insolubilized proteins. Cel- lulosic matrices, as pointed out earlier, exhibit some nonspecific affinity for Coomassie blue but the problem could be overcome by washing the matrices with a mixture of acetic acid, methanol, and water instead of distilled water alone. Various porous, nonporous, or fibrous matrices containing no bound proteins retained very little dye when incubated with the dye reagent and washed with water or the wash solution. The applicability of the assay to proteins associated ionically with insoluble ion exchangers is interesting. Pre- sumably, the proteins get fixed on the support during the incubation with the dye reagent and hence are not eluted during the washing process.

The differences between the protein values obtained by the dye-binding assay and those calculated from the difference in amount of protein added and that remaining after immobilization were, for all the proteins studied, greater than anticipated. Invariably low protein values were obtained by the dye-binding assay (Table 2), indicating that the values calculated by the other procedures are over- estimations. Better agreement obtained between the dye-binding assay and the assay based on iron in the cases of hemoglobin and cytochrome c further substantiates the reliability of the data obtained by the former procedure.

The Coomassie blue assay, as shown in Table 3, could be performed several times on the same sample of immobilized protein. Reliability of the measurement can therefore be increased markedly by repeatedly assaying


the same sample several times. Thus, wastage of the valuable immobilized protein for protein determination can be minimized.

The exact mechanism of binding of Coomassie blue G-250 to protein is not completely known (37). The dye contains two negative charges under acidic conditions of the dye reagent (42). The positively charged side-chain groups of the basic amino acid lysine, and to a smaller extent arginine, have been implicated in the dye-binding process although hydrophobic interactions also play an important role in the binding of the dye to the protein (25). It was also suggested that the dye binding is primarily determined by the amino acid composition in the case of small peptides and by the molecular size in the case of large peptides. Esen (18) reported that the content of basic amino acids is also important for binding of the dye to proteins, in view of his observation that zeins, the proteins lacking lysine and arginine, bind only a quarter of the dye bound by serum albumin. Several immobilization procedures are based on covalent coupling of the side- chain amino groups of proteins to the activated matrix (43,44). Decrease in the dye- binding capacity of proteins becomes likely when such immobilization procedures are employed. Data presented in Fig. 3, however, show that modification of over 70% free amino groups results in less than 10% decrease in the dye-binding capacity of BSA. Evidently not all the amino groups of proteins are involved in the binding of Coomassie blue. Our unpublished observation, that polylysine (Mr 70,000) does not appeciably bind Coomassie blue G-250, supports this observation. It is therefore likely that the amino groups located in the proximity of the hydrophobic pockets are crucial for the dye- binding process. The anionic groups of the dye apparently have a greater possibility of strongly binding to amino groups only after the nonpolar regions of the dye are firmly anchored in the hydrophobic pockets. A highly water-soluble compound like glutar-

aldehyde will, on the other hand, readily react with the amino groups located in the hydrophilic environment. It is also logical to assume that side-chain amino groups of proteins that preferentially react with activated hydrophilic matrices during the immobilization process will be those present in hydrophilic surroundings. In addition, relatively small numbers of the total amino groups are generally involved in covalent coupling of protein with the matrices ( 12,45). Loss in the dye-binding capacities of proteins as a result of immobilization via amino groups is therefore likely to be small. Our preliminary stud- ies indicate that the Coomassie blue assay cannot be applied to enzymes and proteins entrapped in polymeric matrices and proteins immobilized on proteinic supports (46,47). Despite these limitations the Coomassie blue- binding assay described in this article offers several advantages in the quantitation of protein immobilized on several matrices. We have observed that Coomassie blue R-250 can also be used instead of Coomassie blue G-250 for the determination of insolubilized proteins and there was no major difference in the sensitivity of the assays employing either of the dyes. A technique for the quantitation of proteins stained with Coomassie blue G-250 or Coomassie blue R-250 in polyacrylamide gels by the electroelution of the bound dye has been described recently (48). This may be applicable, with some advantage, for the quantitation of protein insolubilized on various matrices using the approach described in this article.

ACKNOWLEDGMENT

Financial assistance was provided to H.A. by CSIR, New Delhi.

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A coomassie blue-binding assay for the microquantitation of immobilized proteins

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